U.S. patent application number 15/565558 was filed with the patent office on 2018-05-03 for method for preparing microencapsulated heat-sensitive bioactive material.
The applicant listed for this patent is HER MAJESTY THE QUEEN IN RIGHT OF CANADA, AS REPRESENTED BY THE MINISTER OF AGRICULTURE AND AGR, HER MAJESTY THE QUEEN IN RIGHT OF CANADA, AS REPRESENTED BY THE MINISTER OF AGRICULTURE AND AGR. Invention is credited to Wuwei CUI, Jianhua GONG, Qian GUO, Huan LIU, Shea MILLER, Qi WANG, Hai YU.
Application Number | 20180119088 15/565558 |
Document ID | / |
Family ID | 57071688 |
Filed Date | 2018-05-03 |
United States Patent
Application |
20180119088 |
Kind Code |
A1 |
WANG; Qi ; et al. |
May 3, 2018 |
METHOD FOR PREPARING MICROENCAPSULATED HEAT-SENSITIVE BIOACTIVE
MATERIAL
Abstract
Microparticles include a matrix of an encapsulating material, in
which are dispersed particles of a low melting point fat and a
bioactive material, such as one or more probiotic bacteria. The
microparticles are formed by preparing an emulsion of melted low
melting point fat in an aqueous mixture of the encapsulating
material, cooling the emulsion below the melting point of the low
melting point fat, dispersing the bioactive material in the
emulsion and spray drying the emulsion. The particles of solid low
melting point fat are believed to protect the bioactive material
from heat damage during the spray drying process.
Inventors: |
WANG; Qi; (Guelph, CA)
; LIU; Huan; (Guelph, CA) ; GONG; Jianhua;
(Guelph, CA) ; YU; Hai; (Guelph, CA) ; GUO;
Qian; (Guelph, CA) ; MILLER; Shea; (Ottawa,
CA) ; CUI; Wuwei; (Guelph, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HER MAJESTY THE QUEEN IN RIGHT OF CANADA, AS REPRESENTED BY THE
MINISTER OF AGRICULTURE AND AGR |
Guelph |
|
CA |
|
|
Family ID: |
57071688 |
Appl. No.: |
15/565558 |
Filed: |
March 24, 2016 |
PCT Filed: |
March 24, 2016 |
PCT NO: |
PCT/CA2016/050344 |
371 Date: |
October 10, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62144137 |
Apr 7, 2015 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23K 40/30 20160501;
A23V 2002/00 20130101; A23P 10/30 20160801; A23V 2002/00 20130101;
A61K 9/1652 20130101; A23L 33/135 20160801; A23V 2002/00 20130101;
A23D 7/05 20130101; A61K 35/747 20130101; A61K 47/42 20130101; A23D
9/05 20130101; A23D 7/0053 20130101; C12N 11/04 20130101; A23V
2250/194 20130101; C12N 1/04 20130101; A23P 10/35 20160801; B01J
13/043 20130101; A23Y 2220/00 20130101; A23V 2200/224 20130101;
A23V 2250/5028 20130101; A23V 2200/3204 20130101; A23V 2250/54246
20130101; A23V 2200/3204 20130101; A23V 2250/194 20130101; A23V
2250/5028 20130101; A23V 2250/54246 20130101; A23V 2200/224
20130101 |
International
Class: |
C12N 1/04 20060101
C12N001/04; B01J 13/04 20060101 B01J013/04; A23P 10/35 20060101
A23P010/35; A23K 40/30 20060101 A23K040/30; A61K 47/42 20060101
A61K047/42 |
Claims
1. A method of preparing microparticles, the method comprising:
heating a low melting point fat above a melting point thereof to
faun a liquid melt; mixing the liquid melt with an aqueous mixture
of an encapsulating material to form an emulsion; cooling the
emulsion below the melting point of the low melting point fat;
dispersing a bioactive material into the emulsion; and spray drying
the emulsion to form the microparticles.
2. The method according to claim 1 wherein the encapsulating
material comprises sodium caseinate.
3. The method according to claim 2 wherein the encapsulating
material further comprises gum arabic.
4. The method according to claim 1 wherein the low melting point
fat has a melting point greater than about 25.degree. C.
5. The method according to claim 4 wherein the low melting point
fat has a melting point of about 25.degree. C. to about 60.degree.
C.
6. The method according to claim 4 wherein the low melting point
fat is selected from shortenings, cocoa butter, margarine, fatty
acids, lard, suet, palm oil, fractionated palm oil, hydrogenated
oils and mixtures thereof.
7. The method according to claim 1 wherein the bioactive material
comprises one or more probiotic bacteria.
8. The method according to claim 7 wherein the one or more
probiotic bacteria comprise one or more Lactobacillus bacteria.
9. Microparticles comprising: a matrix comprising an encapsulating
material; particles of a low melting point fat dispersed in the
matrix of the encapsulating material; and a bioactive material
dispersed in the matrix of the encapsulating material; wherein the
particles of the low melting point fat are substantially separate
and distinct from the bioactive material.
10. The microparticles according to claim 9 wherein the
encapsulating material comprises sodium caseinate.
11. The microparticles according to claim 10 wherein the
encapsulating material further comprises gum arabic.
12. The microparticles according to claim 9 wherein the low melting
point fat has a melting point of greater than about 25.degree.
C.
13. The microparticles according to claim 12 wherein the low point
melting fat has a melting point of about 25.degree. C. to about
60.degree. C.
14. The microparticles according to claim 12 wherein the low
melting point fat is selected from shortenings, cocoa butter,
margarine, fatty acids, lard, suet, palm oil, fractionated palm
oil, hydrogenated oils and mixtures thereof.
15. The microparticles according to claim 9 wherein the bioactive
material comprises one or more probiotic bacteria.
16. The microparticles according to claim 15 wherein the one or
more probiotic bacteria comprise one or more Lactobacillus
bacteria.
Description
BACKGROUND
[0001] The present invention relates to microencapsulation methods
for bioactive materials. More specifically, the present invention
relates to microparticles containing probiotic bacteria or other
heat-sensitive bioactive materials, and to methods of preparing the
microparticles.
[0002] Many bioactive materials, including probiotic bacteria, can
be beneficial to human and animal health when ingested, for
example, as supplements or additives to food products or animal
feed. However, such materials are sensitive to the adverse
environmental conditions encountered when ingested, such as the
acidic conditions found within the stomach, or high bile salt
concentrations found in the upper intestine. Thus, these materials
may undergo significant loss of viability or functionality before
they reach their target site within the body. Encapsulating such
materials can provide protection against such adverse environmental
conditions, thereby improving viability. International Patent
Application WO 2012/077038, U.S. Pat. No. 8,871,266 and US Patent
Application Publications 2012/0189735, 2011/0008493 and
2009/0238885 describe encapsulation of bioactive materials.
[0003] However, bioactive materials can also undergo environmental
challenges during the encapsulation process. For example, spray
drying is a well-established technique for encapsulating food and
feed ingredients. Spray drying is a continuous and rapid process
with low cost and high reproducibility, and thus is highly suitable
for large-scale, industrial applications. However, conventional
spray drying procedures expose bioactive material, such as
probiotic bacterial cells, to adverse conditions, including high
temperature, which can reduce their viability. During spray drying,
bacterial cells experience heat stress, dehydration, oxygen
exposure and osmotic stress, which could lead to the loss of
metabolic activity and even death of the cells. Attempts to address
such challenges include the selection of thermal resistant
bacterial strains, heat treatment of bacteria prior to spray
drying, and the use of prebiotics or thermoprotectants such as
granular starch, soluble fiber and trehalose. However, these
methods can be difficult and time consuming and are not always
successful.
[0004] Therefore, there is a need in the industry for an
alternative method to protect probiotic bacterial cells and other
heat-sensitive bioactive material from damage due to heat exposure
during processing, including encapsulation procedures involving
spray drying. Such a method may make it possible to use spray
drying techniques to conveniently encapsulate heat-sensitive
bioactive materials for which previously known spray drying
processes are not suitable or effective.
SUMMARY
[0005] One aspect of the present invention provides microparticles
including a matrix of an encapsulating material, in which are
dispersed smaller particles of a low melting point fat and a
bioactive material. The particles of the low melting point fat are
substantially separate and distinct from the bioactive
material.
[0006] In another aspect, the present invention provides a method
of preparing microparticles, the method including heating a low
melting point fat to form a liquid melt; mixing the liquid melt
with an aqueous mixture of an encapsulating material to form an
emulsion; cooling the emulsion below the melting point of the low
melting point fat; dispersing a bioactive material into the
emulsion; and spray drying the emulsion to form the
microparticles.
[0007] In at least one embodiment, the encapsulating material
comprises sodium caseinate. In at least one embodiment, the
encapsulating material further comprises gum arabic. In at least
one embodiment, the low melting point fat has a melting point of
greater than about 25.degree. C. In at least one embodiment, the
low melting point fat has a melting point of about 25.degree. C. to
about 60.degree. C. In at least one embodiment, the low melting
point fat is selected from shortenings, cocoa butter, margarine,
fatty acids, lard, suet, palm oil, fractionated palm oil,
hydrogenated oils and mixtures thereof. In at least one embodiment,
the low melting point fat is selected from palm oil, hydrogenated
cottonseed oil and mixtures thereof. In at least one embodiment,
the bioactive material comprises one or more probiotic bacteria. In
at least one embodiment, the one or more probiotic bacteria
comprise one or more Lactobacillus species.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Further features of the present invention will become
apparent from the following written description and the
accompanying figures, in which:
[0009] FIG. 1 is a diagram of a spray-dried microparticle according
to an embodiment of the invention including low melting point fat
particles and probiotic bacteria;
[0010] FIG. 2A is a confocal light microscopy image of a
spray-dried microparticle according to the embodiment of FIG. 1, in
which fat particles appear orange due to selective staining with
Nile Red, bacterial cells are indicated by arrows and appear blue
due to selective staining with DAPI, and sodium caseinate (NaCas)
appears green due to selective staining with FITC;
[0011] FIG. 2B is a confocal light microscopy image of a
spray-dried microparticle similar to the embodiment of FIG. 2A but
containing vegetable oil droplets instead of low melting point fat
particles;
[0012] FIG. 2C is a series of scanning electron micrographs of
spray-dried microparticles according to the embodiment of FIG. 1
containing vegetable oil and sodium caseinate at ratios (w/w) of
0.25:1 (panels a and b), 0.50:1 (panels c and d), 0.75:1 (panels e
and f) or 1:1 (panels g and h) or low melting point fat and sodium
caseinate at ratios (w/w) of 0.25:1 (panels i and j), 0.50:1
(panels k and I), 0.75:1 (panels m and n) or 1:1 (panels o and
p);
[0013] FIG. 3A is a graph showing the thermal stability of
Lactobacillus reuteri K67 in sodium caseinate solution;
[0014] FIG. 3B is a graph showing the thermal stability of
Lactobacillus reuteri S64 in sodium caseinate solution;
[0015] FIG. 3C is a graph showing the thermal stability of
Lactobacillus zeae LB1 in sodium caseinate solution;
[0016] FIG. 4A is a graph showing the survival rate of
Lactobacillus reuteri K67 encapsulated in spray-dried
microparticles including sodium caseinate (NaCas) alone or sodium
caseinate including varying amounts of vegetable oil or low melting
point fat (LMF);
[0017] FIG. 4B is a graph showing the survival rate of
Lactobacillus reuteri S64 encapsulated in spray-dried
microparticles including sodium caseinate (NaCas) alone or sodium
caseinate including varying amounts of vegetable oil or low melting
point fat (LMF);
[0018] FIG. 4C is a graph showing the survival rate of
Lactobacillus zeae LB1 encapsulated in spray-dried microparticles
including sodium caseinate (NaCas) alone or sodium caseinate
including varying amounts of vegetable oil or low melting point fat
(LMF);
[0019] FIG. 5A is a graph showing the survival rate of a fresh
culture of Lactobacillus reuteri K67, or Lactobacillus reuteri K67
encapsulated in spray-dried microparticles including sodium
caseinate (NaCas) alone or sodium caseinate including varying
amounts of vegetable oil or low melting point fat (LMF), on MRS
agar supplemented with 5% NaCl;
[0020] FIG. 5B is a graph showing the survival rate of a fresh
culture of Lactobacillus zeae LB1, or Lactobacillus zeae LB1
encapsulated in spray-dried microparticles including sodium
caseinate (NaCas) alone or sodium caseinate including varying
amounts of vegetable oil or low melting point fat (LMF), on MRS
agar supplemented with 5% NaCl;
[0021] FIG. 5C is a graph showing the survival rate (on a
logarithmic scale) of a fresh culture of Lactobacillus reuteri S64,
or Lactobacillus reuteri S64 encapsulated in spray-dried
microparticles including sodium caseinate (NaCas) alone or sodium
caseinate including varying amounts of vegetable oil or low melting
point fat (LMF), on MRS agar supplemented with 5% NaCl;
[0022] FIG. 6A is a graph showing the survival rate of
Lactobacillus zeae LB1 encapsulated in spray-dried microparticles
including varying proportions of sodium caseinate (NaCas) and gum
Arabic in addition to low melting point fat, compared to
unencapsulated (free) LB1, when exposed to simulated gastric fluid
(1 h - 2 h) and simulated intestinal fluid (3 h-6 h);
[0023] FIG. 6B is a graph showing the release of Lactobacillus zeae
LB1 from the spray-dried microparticles of FIG. 6A when exposed to
simulated gastric fluid (1 h-2 h) and simulated intestinal fluid (3
h-6 h);
[0024] FIG. 7 is a graph showing the survival rate of Lactobacillus
zeae LB1 from the spray-dried microparticles of FIG. 6A after
storage at 4.degree. C. for varying lengths of time;
[0025] FIG. 8A is a graph showing the effect of outlet temperature
on the water content of spray dried microparticles including
Lactobacillus zeae LB1 and either sodium caseinate (NaCas) alone or
a 1:1 ratio (w/w) of sodium caseinate and either vegetable oil or
low melting point fat (LMF);
[0026] FIG. 8B is a graph showing a plot of the survival rate of
Lactobacillus zeae LB1 in the spray dried microparticles of FIG. 8A
vs. water content;
[0027] FIG. 8C is a graph showing the effect of outlet temperature
on the water activity of the spray dried microparticles of FIG.
8A;
[0028] FIG. 8D is a graph showing a plot of the survival rate of
Lactobacillus zeae LB1 in the spray dried microparticles of FIG. 8A
vs. water activity;
[0029] FIG. 8E is a graph showing a plot of the water activity of
the spray dried microparticles of FIG. 8A vs. water content;
[0030] FIG. 9A is a graph showing a series of differential scanning
calorimetry (DSC) curves of emulsions of vegetable oil in 10% (w/w)
aqueous sodium caseinate (NaCas) solution at various ratios of oil
to NaCas; the control is 10% (w/w) aqueous NaCas solution
containing no oil;
[0031] FIG. 9B is a graph showing a series of differential scanning
calorimetry (DSC) curves of emulsions of low melting point fat
(LMF) in 10% (w/w) aqueous sodium caseinate (NaCas) solution at
various ratios of LMF to NaCas; the control is 10% (w/w) aqueous
NaCas solution containing no LMF; and pure fat represents
unemulsified LMF; and
[0032] FIG. 10 is a graph of a plot of survival of Lactobacillus
zeae LB1 encapsulated in spray-dried microparticles including
sodium caseinate (NaCas) including varying amounts of low melting
point fat (LMF) vs. the melting enthalpy of emulsions of LMF in 10%
(w/w) aqueous NaCas solution containing corresponding ratios by
weight of LMF to NaCas.
DETAILED DESCRIPTION
[0033] One aspect of the present invention provides microparticles.
With reference to FIG. 1, spray dried microparticles 10 include a
matrix 12 of an encapsulating material, dispersed in which are
smaller particles of a low melting point fat 14 and a bioactive
material 16. The particles of the low melting point fat are
substantially separate and distinct from the bioactive material. As
used herein, the term "microparticle" is intended to mean a
particle which has a diameter of from about 0.1 .mu.m to about 100
.mu.m.
[0034] As used herein, the term "about" or "approximately" as
applied to a numerical value or range of values is intended to mean
that the recited values can vary within an acceptable degree of
error for the quantity measured given the nature or precision of
the measurements, such that the variation is considered in the art
as equivalent to the recited values and provides the same function
or result. For example, the degree of error can be indicated by the
number of significant figures provided for the measurement, as is
understood in the art, and includes but is not limited to a
variation of .+-.1 in the most precise significant figure reported
for the measurement. Typical exemplary degrees of error are within
20 percent (%), preferably within 10%, and more preferably within
5% of a given value or range of values. Alternatively, and
particularly in biological systems, the terms "about" and
"approximately" can mean values that are within an order of
magnitude, preferably within 5-fold and more preferably within
2-fold of a given value. Numerical quantities given herein are
approximate unless stated otherwise, meaning that the term "about"
or "approximately" can be inferred when not expressly stated.
[0035] As used herein, the term "substantially" refers to the
complete or nearly complete extent or degree of an action,
characteristic, property, state, structure, item, or result. For
example, a particle that is "substantially" separate from another
particle within a matrix is intended to mean that the particles are
either completely separated by intervening matrix material or
nearly completely separated so that some incidental contact is
possible, but the particles do not undergo any degree of contact or
intermixing which would have a measureable effect on their
individual functions or structures. The exact allowable degree of
deviation from absolute completeness may in some cases depend on
the specific context. However, generally speaking the nearness of
completion will be so as to have the same overall result as if
absolute and total completion were obtained.
[0036] The use of "substantially" is equally applicable when used
in a negative connotation to refer to the complete or near complete
lack of an action, characteristic, property, state, structure,
item, or result. For example, a composition that is "substantially
free of" particles would either completely lack particles, or so
nearly completely lack particles that the effect would be the same
as if it completely lacked particles. In other words, a composition
that is "substantially free of" an ingredient or element may still
actually contain such item as long as there is no measurable effect
thereof.
[0037] As used herein, the term "bioactive material" is intended to
mean microorganisms, material derived from or produced by organisms
or microorganisms (including but not limited to tissue, genetic
material, extracts, products including but not limited to enzymes,
and the like), or organic material which has biological activity or
which is necessary or desirable to sustain life functions
(including but not limited to drugs, food and organic nutrients
including but not limited to proteins, carbohydrates, vitamins, and
the like). As used herein, the term "microorganisms" is intended to
mean unicellular, multicellular or non-cellular microscopic
organisms and includes but is not limited to prokaryotic
microorganisms including but not limited to bacteria, archaea and
the like; eukaryotic microorganisms including but not limited to
algae, protists, fungi, yeasts, molds, mites, nematodes and the
like; and infectious particles including but not limited to
viruses, phages, prions and the like. Bioactive material can be,
but need not necessarily be, alive.
[0038] In at least one embodiment, the bioactive material is a
heat-sensitive bioactive material whose viability can be reduced if
the bioactive material is exposed to temperatures above a
predefined range. As used herein, the term "viability" is intended
to mean the ability to live or be sustained, or to fulfil a
biological function. Conditions under which a bioactive material is
viable need not be those under which the bioactive material is
actively growing or functioning, but can also include conditions
under which the bioactive material is inactive or dormant, as long
as it retains at least some potential to live or fulfil its
function. Non-living bioactive material can have viability if it
has not decomposed or been deactivated beyond its ability to fulfil
its intended biological function.
[0039] It will be understood by the skilled person that different
bioactive materials have different optimal temperature ranges at
which viability can be maintained or preserved. Therefore some
bioactive materials are readily damaged or destroyed, so as to lose
or experience reduced viability, by exposure to temperatures at
which other bioactive materials will retain full or significant
viability. For example, damage can occur at various sites in
bacterial cells including the cell wall, cytoplasmic membrane,
ribosomes, RNA and DNA. However, there is often a critical
temperature above which the survival of cells decreases
dramatically, and this critical temperature can be different for
different microorganisms, including but not limited to different
species, strains, varieties or isolates. At temperatures below the
critical temperature, the cell membrane is likely to be the main
site at which damage occurs, while at temperatures above the
critical temperature, denaturation of ribosomes and/or proteins, as
well as damage to the cell wall can occur and lead to thermal death
of the cells.
[0040] The present microparticles include a matrix of an
encapsulating material. Suitable encapsulating materials are well
known in the art and include, but are not limited to, proteins such
as casein or sodium caseinate, whey protein, soy protein, gelatin
and the like, carbohydrates such as gum arabic, carrageenan, locust
bean gum, gellan gum, xanthan gum, cellulose acetate phthalate,
starch, pectin, alginate, chitosan and the like, and mixtures
thereof.
[0041] The present microparticles include particles of a low
melting point fat dispersed in the matrix of the encapsulating
material. In at least one embodiment, the low melting point fat
will have a melting point above normal room temperature, so as to
be in the solid phase under normal ambient conditions. Thus, a low
melting point fat would not include an oil which is normally liquid
under normal ambient conditions, as understood in the art. In at
least one embodiment, the low melting point fat will have a melting
point above about 25.degree. C. In at least one embodiment, the low
melting point fat will have a melting point in the range of about
25.degree. C. to about 60.degree. C. In at least one embodiment,
the low melting point fat will have a melting point in the range of
about 25.degree. C. to about 45.degree. C. In at least one
embodiment, the low melting point fat will have a melting point in
the range of about 30.degree. C. to about 45.degree. C. Suitable
low melting point fats are known and include but are not limited to
shortenings, cocoa butter, margarine, fatty acids, lard, suet, palm
oil, fractionated palm oil, hydrogenated oils and mixtures thereof.
Hydrogenated oils include but are not limited to hydrogenated palm
oil, hydrogenated cottonseed oil and hydrogenated coconut oil. In
at least one embodiment, the low melting point fat is selected from
palm oil, hydrogenated cottonseed oil and mixtures thereof.
[0042] In at least one embodiment, the ratio of low melting point
fat to encapsulating material in the microparticles varies from
about 0.25:1 to about 1:1 by weight. In at least one embodiment,
the ratio of low melting point fat to encapsulating material in the
microparticles varies from about 0.50:1 to about 1:1 by weight. In
at least one embodiment, the ratio of low melting point fat to
encapsulating material in the microparticles varies from about
0.75:1 to about 1:1 by weight. In at least one embodiment, the
ratio of low melting point fat to encapsulating material in the
microparticles is about 1:1 by weight.
[0043] In at least one embodiment, the present microparticles are
prepared by heating the low melting point fat to form a liquid
melt; mixing the liquid melt with an aqueous mixture of an
encapsulating material to form an emulsion; cooling the emulsion
below the melting point of the low melting point fat to allow
solidification of the fat particles, dispersing the bioactive
material into the emulsion; and spray drying the emulsion to form
the microparticles.
[0044] The low melting point fat can be melted to form the liquid
melt at any temperature above its melting point which will maintain
the low melting point fat in liquid form without causing measurable
or detrimental decomposition. Once melted, the liquid melt can be
mixed with an aqueous mixture of an encapsulating material at a
temperature at which the low melting point fat would remain melted,
so as to form an emulsion. The emulsion can be prepared by using
techniques well known in the art, including but not limited to
blending and/or homogenizing the mixture of the liquid melt and the
aqueous mixture of the encapsulating material, and treating the
mixture of the liquid melt and the aqueous mixture of the
encapsulating material with ultrasound.
[0045] In at least one embodiment, the aqueous mixture of the
encapsulating material is an aqueous solution of the encapsulating
materials described herein above. In at least one embodiment, the
aqueous mixture further comprises one or more additives, including
but not limited to prebiotics and protectants and antioxidants.
Suitable prebiotics and protectants include but are not limited to
sugars, oligosaccharides and polysaccharides, including but not
limited to starch, maltodextrin, inulin, trehalose, and the like.
Suitable antioxidants are advantageously lipid-soluble
antioxidants, including but not limited to butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone
(TBHQ), vitamin E, tocopherols, tocotrienols, and the like.
[0046] In at least one embodiment, the prepared emulsion of the low
melting point fat and the aqueous mixture of the encapsulating
material is cooled below the melting point of the low melting point
fat, such that solid particles of the low melting point fat are
formed, and the bioactive material is dispersed in the emulsion.
The bioactive material can be added to the emulsion in any
convenient form, including but not limited to a solution or
dispersion in a suitable solvent, such as water. If the bioactive
material includes one or more microorganisms, it can be added as a
suspension in a culture medium or diluted culture medium. The
bioactive material can be dispersed in the emulsion by any known
technique, including but not limited to stirring and vibration.
[0047] The emulsion containing the dispersed bioactive material is
then spray dried, using apparatus and conditions well known in the
art, to form the encapsulated bioactive material in the form of a
powder. Advantageously, the outlet temperature of the spray drying
apparatus is as high as possible without causing deleterious effect
to the bioactive material, as will be understood in the art.
Without being bound by theory, it is believed that higher outlet
temperatures, where possible without deleterious effect, will
advantageously reduce the water content of the spray dried powder
and improve the storage stability of the spray dried powder. In at
least one embodiment, the spray drying can be carried out at an
outlet temperature of from about 65.degree. C. to about 80.degree.
C. However, the skilled person is readily able to select other
suitable outlet temperatures for various bioactive materials in
light of the teaching herein.
[0048] Without being bound by theory, it is believed that the
droplets of low melting point fat dispersed through the emulsion
return to the solid crystal phase when the emulsion is cooled below
the melting point of the low melting point fat. After the emulsion
containing the bioactive material is transferred into the spray
drier, it first passes for a short period through a chamber in
which the temperature is almost as high as the inlet temperature.
The emulsion is then sprayed through a nozzle as micro-droplets
into the drying chamber. The encapsulated bioactive material can be
exposed to high temperatures in these locations. At such
temperatures, the low melting point fat particles can melt or
undergo a solid to liquid phase transition, thereby absorbing heat
while maintaining a constant temperature. Because of this heat
absorption, the temperature of any bioactive material embedded in
the encapsulating material in the vicinity of the melting fat
particles is prevented from increasing to the extent that it would
if the fat particles were not present. Thus, in at least one
embodiment of the present microparticles, the particles of low
melting point fat within the matrix of encapsulating material can
protect the encapsulated bioactive material, including but not
limited to probiotic bacteria, from heat damage during the spray
drying process. Furthermore, in at least one embodiment of the
present microparticles, it is contemplated that the particles of
low melting point fat within the matrix of encapsulating material
can protect the encapsulated bioactive material, including but not
limited to probiotic bacteria, from heat damage during other
processing steps involving heat.
EXAMPLES
[0049] Other features of the present invention will become apparent
from the following non-limiting examples which illustrate, by way
of example, the principles of the invention.
[0050] Sodium caseinate (NaCas) was purchased from Sigma-Aldrich
Chemical Co., Ltd (St. Louis, Mo., USA). Vegetable oil and low
melting point fat (LMF) were obtained from UNICO Inc. (ON, Canada)
and 101 Loders Croklaan Inc. (Sanslrans.TM. 39, IL, USA),
respectively. Glassware was sterilized at 121.degree. C. for 15
min. The stains 4',6-diamidino-2-phenylindole (DAPI) and
fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich
(St-Louis, Mo., USA), and
9-diethylamino-5H-benzo[a]phenoxazine-5-one (Nile Red) was
purchased from Kodak (Rochester, N.Y., USA).
[0051] The results of each data point in the graphs shown in the
Figures represent the mean of triplicate experiments and the error
bars indicate the standard deviations for the data points. All
differences were considered statistically significant at a
0.05.
Example 1
Preparation of Lactobacillus isolates
[0052] Lactobacillus zeae LB1 (LB1) and Lactobacillus reuteri S64
(S64) and K67 (K67) are isolates from chicken or pig intestines
with the capacity to inhibit Salmonella or E. coli infection in
Caenorhabditis elegans, broiler chickens, or pigs. Isolates from
stock cultures in 15% (v/v) aqueous glycerol at -80.degree. C. were
cultured on de Man, Rogosa and Sharpe (MRS) agar (BD Institution,
MD, USA) for recovery and single colony purification. Each isolate
was sub-cultured twice in MRS broth at 37.degree. C. for 24 h prior
to preparation of a fresh culture inoculated (1%, v/v) in MRS broth
and grown at 37.degree. C. for 12 hours. All cultures were grown
under anaerobic atmosphere (80% N.sub.2, 15% CO.sub.2 and 5%
H.sub.2) and were harvested in the early stationary phase. A
probiotic culture in the stationary phase often has better heat
resistance than in the exponential phase. Bacterial cells were
harvested by centrifugation (Sorvall.TM. RC 6 Plus, Thermo
Scientific Inc., MA, USA) at 4,000.times. g for 20 min (4.degree.
C.) and washed twice with sterile 0.85% (w/v) sodium chloride
solution. The pellet was then re-suspended in sterile 0.85% (w/v)
sodium chloride solution to obtain a suspension containing
approximately 10.sup.10 colony-forming units (CFU)/mL. The
bacterial suspension (10.sup.10 CFU/mL) was stored at 4.degree. C.
and used on the same day.
Example 2
Thermal tolerance of Lactobacillus isolates
[0053] Two 50 mL bottles containing 19 mL NaCas solution (10%, w/w)
were placed in a water bath at test temperatures of 54.degree. C.,
57.degree. C., 60.degree. C., 63.degree. C. and 66.degree. C. One
of the bottles was a control used to monitor the temperature. When
the desired temperature was reached, 1 mL of either Lactobacillus
zeae LB1 (LB1) or Lactobacillus reuteri S64 (S64) or K67 (K67) cell
suspension (Example 1) was added to the second bottle. At selected
intervals (between 30 s and 5 min), 1 mL aliquots were removed from
the test bottle, serially diluted in MRS broth and plated on MRS
agar for CFU counts. Enumeration was performed after 24 h of
anaerobic incubation at 37.degree. C. The plating and enumeration
were accomplished using an Eddy Jet Spiral Plater (Neu-tec Group,
Farmingdale, N.Y., USA).
[0054] Results
[0055] The heat tolerance of the three Lactobacillus isolates is
shown in FIGS. 3A (K67), 3B (S64) and 3C (LB1). The viability of
the three isolates was unchanged at 54.degree. C. for up to 5 min.
At 57.degree. C., a decrease of 0.55 log CFU mL.sup.-1 was obtained
for LB1, while the other two isolates showed no decrease in
viability up to 5 min. At 60.degree. C., LB1 and K67 experienced
decreases of 2.5 log CFU mL.sup.-1 and 0.35 log CFU mL.sup.-1,
respectively, but no significant change was observed for S64. These
results suggest that for each isolate, there is a critical
temperature (60.degree. C. for LB1, 63.degree. C. for S64 and K67)
above which survival decreases dramatically.
[0056] The D-values, or the time required to kill 90% of the cells
at various temperatures, of the three different probiotic strains
are presented in Table 1. D-values can be used as an indicator of
the heat tolerance of microorganisms, such that the greater the
D-value, the better the heat tolerance.
TABLE-US-00001 TABLE 1 Temperature D-value (min) (.degree. C.) LB1
K67 S64 54 333.3 212.3 333.3 57 12.3 62.5 88.1 60 3.1 18.2 44.6 63
2.2 3.8 8.2 66 1.3 2.8 3.1
[0057] Relatively high D-values were found for all three strains at
temperatures below 57.degree. C. Among the three isolates, the
D-value of S64 was greater than those of LB1 and K67 at all
temperatures investigated, indicating that S64 has the best thermal
tolerance, while LB1 shows the poorest.
Example 3
Microencapsulation of Lactobacillus isolates
[0058] Sodium Caseinate Microencapsulation
[0059] Low melting point fat (LMF) was preheated at 50.degree. C.
in a water bath to melt all crystals. Vegetable oil or LMF was then
added into 100 mL aqueous sodium caseinate (NaCas) solution (10%
w/w, 40.degree. C.) with varying ratios of lipid to NaCas
(0.25:1.00, 0.50:1.00, and 1.00:1.00 w/w). NaCas solution without
vegetable oil or LMF (0:1.00 w/w) was used as a control. The
mixtures were coarsely mixed using a blender (Polytron.RTM. PT
10-35 GT-D, Kinematica Corporation, Switzerland) at 6000 rpm for 1
min (40.degree. C.) and then recirculated three times through a
high pressure homogenizer (Nano DeBEE, B.E.E. International Inc.,
MA, USA) at 3000 psi (40.degree. C.). The prepared emulsions were
left at 0.degree. C. overnight, and Lactobacillus cultures
(Lactobacillus reuteri K67 (K67) or S64 (S64) or Lactobacillus zeae
LB1 (LB1)) were dispersed into the emulsions and stirred at 100 rpm
for 10 min at 0.degree. C. The final mixtures (10.sup.9 CFU/g dry
coating material) were then spray dried in a laboratory scale spray
dryer (ADL 310, Yamato Scientific America Inc., CA, USA), at a
constant inlet temperature of 170.degree. C. and outlet temperature
of 80.degree. C. and a flow rate of 5 mL/min. Dried powder samples
were collected from the base of the cyclone and stored in tightly
sealed sterile bottles at 4.degree. C.
[0060] Sodium Caseinate-gum Arabic Microencapsulation
[0061] Sodium caseinate (NaCas)-gum arabic (GA) complex solutions
having ratios of NaCas: GA of 4:0, 3:1, 2:2, 1:3, 0:4 (w/w) (total
solid content 10% (w/w)) were prepared in distilled water and
stirred overnight at 4.degree. C. The solutions were adjusted to pH
7.0 and pre-heated to 40.degree. C. LMF was then added into the
complex solutions at a ratio of 1:1 (w/w). Emulsification,
dispersion of Lactobacillus cultures into the emulsions and spray
drying were carried out as described above.
Example 4
Surface and Internal Microstructure of the Spray Dried
Microparticles
[0062] Confocal Laser-scanning Microscopy (CLSM)
[0063] Microparticles were rehydrated on a glass slide with a drop
of triple fluorescent stain (4',6-diamidino-2-phenylindole (DAPI)
0.0005% (w/v), fluorescein isothiocyanate (FITC) 0.0007% (w/v) and
9-diethylamino-5H-benzo[.alpha.]phenoxazine-5-one (Nile Red) 0.15%
(w/v) in a 100 mM CaCl2 solution). A cover slip was then applied
with 4 drops of nail polish in the corners as a spacer to prevent
compression of the microparticles. Lipid particles appear orange
due to selective staining with Nile Red, bacterial cells appear
blue due to selective staining with DAPI and sodium caseinate
(NaCas) appears green due to selective staining with FITC.
Observations of bacterial cells, protein and lipid were performed
with a Carl Zeiss LSM 510 Duo confocal laser-scanning microscope
(Gottingen, Germany) using excitation lines at 405, 488 and 532 nm
and emission band pass 420-490 nm, 515-550 nm and 575-700 nm for
DAPI, FITC and Nile Red respectively.
[0064] Results
[0065] As seen in FIGS. 2A and 2B, lipid particles (orange) and
bacterial cells (blue, indicated by arrows) were dispersed
throughout the NaCas matrix (green) with no visible differences
between the oil and fat containing samples. With increasing core
(lipid) to wall (NaCas) ratio, the density of the oil/fat globules
within the particles increased, but the diameter of the oil/fat
globules remained constant, possibly because the same process and
parameters were applied during the preparation of emulsions and
spray drying. Bacterial cells were observed only in the NaCas
matrix within the microparticles, and not within the fat particles
or oil droplets, reflecting the overall hydrophilic nature of the
bacteria surfaces. When mixed with the emulsion, the bacteria are
believed to spontaneously move into the hydrophilic phase (NaCas
matrix) instead of the hydrophobic phase (oil or fat phase).
[0066] Scanning Electron Microscopy (SEM)
[0067] The surface morphology of the microparticles was observed
with a scanning electron microscope at an accelerating voltage of
20 kV. Prior to recording microscopic observations, carbon sticky
tabs were attached to aluminum stubs and the sticky surface was
lightly coated with gold for 45 seconds to help reduce charging in
the microscope. Small amounts of microparticles were then dusted
onto the stubs, spread with a spatula, and the excess particles
were blown off with forced air. The stubs were then coated with
gold for 2.5 minutes, for a final gold thickness of approximately
8.9 nm.
[0068] Results
[0069] Scanning electron micrographs are presented in FIG. 2C of
microparticles produced with varying ratios of oil (panels a to h)
or low melting point fat (panels i to p) to sodium caseinate. The
diameters of spray dried microparticles were around 15 to 20 .mu.m
and no bacteria were observed on the surface of the microparticles.
The microparticles containing different lipid core materials (oil
or low melting point fat) were similar in appearance, indicating
that the lipid used did not affect the morphology of the particles.
The shape of the particles varied from irregular to spherical, and
the surfaces of the particles were mostly wrinkled with concavities
which is believed to be attributed to the shrinkage of the
particles caused by rapid evaporation of the water.
Example 5
Survival of Spray Dried Microencapsulated Lactobacillus
Isolates
[0070] Bacterial cell viability of spray dried powders (Example 3)
was determined by the standard plate counting method. Spray dried
powders (0.5 g) were dispersed in 4.5 mL 0.2 M phosphate buffer (pH
7.0) and homogenized for 1 min at 4000 rpm (Polytron.RTM. PT 10-35
GT-D, Kinematica Corporation, Switzerland). Enumeration of cells
was carried out by plating on MRS agar. Colony forming units (CFU)
were enumerated manually after incubation at 37.degree. C. for 24
h.
survival rate ( % ) = C F U / g spray dried powder C F U / g total
solid in initial solution prior to spray drying .times. 100 %
##EQU00001##
[0071] Results
[0072] Among the control samples of the three Lactobacillus
isolates (i.e. those containing NaCas but no oil or LMF), the
highest survival rate (.about.95%) was obtained with Lactobacillus
reuteri S64 (S64), as seen in FIG. 4B, which is consistent with the
higher thermal tolerance of this isolate as noted in Example 2
above. Addition of either vegetable oil or LMF did not alter the
survival rates of Lactobacillus reuteri K67 (K67) (FIG. 4A) and S64
(FIG. 4B) after spray drying. However, as seen in FIG. 4C, the
survival rate of Lactobacillus zeae LB1 (LB1) in the control sample
was only about 16%. Among the samples of LB1 containing vegetable
oil as core material, the survival rates were almost the same
(around 16%), and not significantly different from that in the
control sample (p<0.05). In contrast, addition of LMF increased
the survival rate of LB1 from 16% to 63% as the LMF to wall ratio
increased from 0.25 to 1.00.
Example 6
Salt Tolerance of Microencapsulated Lactobacillus Isolates
[0073] Fresh cultures and spray dried microparticles prepared as
described in Example 3 of Lactobacillus reuteri K67 (K67),
Lactobacillus zeae LB1 (LB1) and Lactobacillus reuteri S64 (S64)
were plated on MRS agar without NaCl or supplemented with NaCl (5%,
w/v). The plates were incubated for up to 3 days under anaerobic
conditions and viable numbers were recorded. The survival rate was
determined using the following equation:
Survival rate ( % ) = N s N n ##EQU00002##
where N.sub.s and N.sub.n represent the survival number grown on
MRS agar containing NaCl and MRS agar without NaCl,
respectively.
[0074] The sensitivity of bacteria to salt was defined as
follows:
Sensitivity (%)=100 (%)-Survival rate (%)
[0075] Results
[0076] Fresh cultures of the three isolates exhibited varying
degrees of tolerance to salt, with survival rates of 96%, 76%, and
5% for K67, LB1 and S64, respectively, as seen in FIGS. 5A, 5B, and
5C, respectively. The survival rates on NaCl-MRS agar of all spray
dried bacterial isolates encapsulated in NaCas without inclusion of
oil or LMF were markedly lower than those of the fresh bacterial
cultures: 30%, 5%, and 0.2% for K67, LB1 and S64, respectively. For
isolates K67 and S64, spray drying induced minimal loss in cell
viability (Example 5), but resulted in a significant decrease in
salt tolerance. This result suggests that although the bacterial
cells survived the spray drying process, some damage to the cell
membrane may have occurred, so that the tolerance to salt
decreased. In the case of LB1, severe loss of viability was
observed after spray drying (Example 5, FIG. 4C), accompanied by
further loss of salt tolerance (FIG. 5B), which suggests that the
cell damage may be more extensive.
[0077] The survival rates on NaCl-supplemented MRS agar of all
three isolates microencapsulated with vegetable oil were similar to
those in the control NaCas-only microparticles, suggesting that
inclusion of oil in the formula did not affect the salt tolerance
of bacteria after spray drying. In contrast, significant increases
in survival rate on NaCl-supplemented MRS agar were observed for
isolates LB1 and K67 in the presence of LMF when the ratio of LMF
to wall material reached 1.0 (P<0.05), indicating that the
addition of LMF to the microparticles can protect these isolates
against damage experienced during spray drying which would have
otherwise been expected to further decrease their tolerance to
salt. The presence of LMF in microparticles of encapsulated S64 had
little effect on the salt tolerance of the relatively thermally
tolerant (Example 2, FIG. 3B; Example 5, FIG. 4B) but highly salt
intolerant (FIG. 5C) S64 isolate.
Example 7
Survival and Release of Microencapsulated Lactobacillus zeae LB1
(LB1) Under Simulated Gastrointestinal Conditions
[0078] Microparticles (Example 3, 0.1 g) containing Lactobacillus
zeae LB1 (LB1) encapsulated in a matrix containing varying
proportions of sodium caseinate (NaCas) and gum Arabic, and
containing low melting point fat in a 1:1 ratio by weight with the
encapsulating matrix (Example 3), or free LB1 bacterial cells
harvested as described in Example 1 and diluted in sterile 0.85%
(w/v) sodium chloride solution to .about.10.sup.9 CFU/mL (0.1 mL),
were added to test tubes containing 9.9 mL of pre-warmed
(37.degree. C.) freshly prepared and filter sterilized simulated
gastric fluid (SGF) (0.32 wt% pepsin, 0.2 wt% NaCl, adjusted to pH
2.0 with 1M HCl). The samples were vortexed and incubated at
37.degree. C. Samples were removed at 30, 60, 90, and 120 min for
bacterial counting, and the pH was then rapidly adjusted to 7.0
with 1M NaOH. Simulated intestinal fluid (SIF) (pancreatin (10 g/L)
and bile salts (8 g/L) in phosphate buffer (0.2M, pH=7.0)) (10 mL)
was added, and 1 mL aliquots were removed from each sample for
bacterial counting after exposure to SIF for a further 1, 2, 3, and
4 h.
[0079] For the measurement of protection properties of
microparticles, samples (1 mL) were added to 9 mL phosphate
buffered saline (PBS) and homogenized for 1 min at 4000 rpm before
determination of viable cell numbers. For the measurement of
release properties of microparticles, samples (1 mL) were withdrawn
without homogenization and directly added into 9 mL PBS for
bacterial counting. Enumeration of cells was carried out by plating
on MRS agar. Colony forming units (CFU) were enumerated manually
after incubation at 37.degree. C. for 24 h.
[0080] Results
[0081] Survival of encapsulated LB1 during simulated
gastrointestinal digestion (2 hours of exposure to SGF, followed by
4 hours of exposure to SIF) is shown in FIG. 6A. Free cells died
very quickly and no viable bacterial cells were detected after 1 h
in SGF. However, for encapsulated bacteria samples (NaCas with or
without gum arabic (GA)), the survival increased significantly.
Among these samples, survival rates of bacteria microencapsulated
with only NaCas or gum arabic were similar to each other and lower
than that of other encapsulated samples. As the gum arabic content
in the wall material increased (from a NaCas: GA ratio of 3:1 to
1:3), the survival of encapsulated bacteria increased, with a loss
of viability of only 1.2 log over 6 h of test time for the sample
having a NaCas: GA ratio of 1:3.
[0082] Release of encapsulated LB1 during simulated
gastrointestinal digestion is shown in FIG. 6B. Free cells died
very quickly and no live bacteria were detected after 1 h in SGF.
For the encapsulated samples, the number of viable cells released
from the microparticles remained constant (0-1 log CFU/g) during
the first two hours of exposure to SGF and increased significantly
when exposed to SIF. All the viable bacteria in the microparticles
were released within 1 h when exposed to SIF.
Example 8
Storage Stability of Microencapsulated Lactobacillus zeae LB1
(LB1)
[0083] Samples of spray dried microparticles containing
Lactobacillus zeae LB1 (LB1) encapsulated in a matrix containing
varying proportions of sodium caseinate (NaCas) and gum arabic, and
containing low melting point fat in a 1:1 ratio by weight with the
encapsulating matrix (Example 3) were stored at 4.degree. C. in
sealed polyethylene bags placed in sealed glass bottles. Samples
were removed at 1 week intervals for determination of viable
bacterial count by the standard plate counting method described in
Example 5.
[0084] Results
[0085] As seen in FIG. 7, minimal reduction in bacterial count was
seen during the first 4 weeks of storage at 4.degree. C. Even after
storage for 16 weeks, less than 1 log reduction in viability was
observed.
Example 9
Water Content and Water Activity of Spray Dried Microparticles
[0086] Spray dried microparticles of Lactobacillus zeae LB1 (LB1)
were prepared from sodium caseinate (NaCas) alone or mixed with
vegetable oil in a 1:1 ratio (w/w) as described in Example 3. Spray
drying was carried out at outlet temperatures of 65.degree. C.,
70.degree. C., 75.degree. C. or 80.degree. C. A comparison sample
of spray dried microparticles of LB1 was prepared from a 1:1 ratio
(w/w) of NaCas and LMF at an outlet temperature of 80.degree.
C.
[0087] Weighing dishes were dried in an oven (105.degree. C.) to a
constant weight and then cooled in a desiccator containing silica
gel. The weight of the empty dish was recorded (a), approximately 3
g of powder was added, and the dish was weighed again (b). The
loaded dish was placed in the oven at 105.degree. C. for 24 h, then
cooled to room temperature in a desiccator and weighed again (c).
The heating and cooling process was repeated until the weight (c)
was constant. The water content was calculated as:
Water content = ( b - c ) .times. 100 % ( c - a ) ##EQU00003##
where a is the weight of the empty dish; b is the weight of the
dish and the wet powder; and c is the weight of the dish and the
dried powder.
[0088] The water activity was measured at 25.degree. C. using a
water activity meter (Aqualab 4TE, Decagon Devices Inc., USA).
[0089] Results
[0090] Microparticles spray dried at 80.degree. C. and containing
NaCas only were found to have a water content of 6.80% by weight;
whereas microparticles spray dried at 80.degree. C. and formulated
with a 1:1 ratio of oil:NaCas or LMF:NaCas were found to have a
water content of 3.25% by weight and 3.68% by weight, respectively.
Assuming that the water is present in the NaCas phase only and is
substantially absent from the lipid phase, the water content of the
NaCas phase of the microparticles formulated with a 1:1 ratio of
oil:NaCas or LMF:NaCas would be 6.78% by weight and 7.67% by
weight, respectively. As seen in FIGS. 2A and 2B, the LB1 cells are
primarily located in the NaCas phase of the microparticles.
[0091] To determine whether the relatively high water content in
the NaCas phase of the 1:1 LMF:NaCas microparticles could have
partially contributed to the high survival of bacteria in these
microparticles, microparticles having similar water content but
containing either NaCas alone or 1:1 oil:NaCas were prepared by
spray drying at various outlet temperatures. As seen from the data
presented in FIG. 8A, an outlet temperature of about 74.degree. C.
would be required to provide microparticles containing either NaCas
alone or 1:1 oil:NaCas which would have a water content of about
7.6%, similar to that found in 1:1 LMF:NaCas microparticles spray
dried at 80.degree. C. As can be seen from the data presented in
FIG. 8B, the interpolated survival rate of LB1 would be similar in
microparticles containing either NaCas alone or 1:1 oil:NaCas and
having a water content of about 7.6%.However, the interpolated
survival rate of LB1 in microparticles containing either NaCas
alone or 1:1 oil:NaCas would be much lower than the survival rate
observed for LB1 encapsulated in 1:1 LMF:NaCas microparticles spray
dried at 80.degree. C. and having a similar water content. This
data thus indicates that the water content of the microparticles is
not primarily responsible for the improved survival rate of LB1
cells in microparticles containing LMF particles.
[0092] Microparticles spray dried at 80.degree. C. and containing
NaCas only were found to have a water activity of 0.18; whereas
microparticles spray dried at 80.degree. C. and formulated with a
1:1 ratio of oil:NaCas or LMF:NaCas were found to have a water
activity of 0.19 and 0.20, respectively. As seen from the data
presented in FIG. 8C, the water activity of microparticles
formulated with NaCas only and spray dried at an outlet temperature
of 75.degree. C. and the water activity of microparticles
formulated with 1:1 ratio of oil:NaCas and spray dried at an outlet
temperature of 72.degree. C. would be expected to be similar to the
water content of microparticles formulated with a 1:1 ratio of
LMF:NaCas and spray dried at 80.degree. C. However, as seen from
the data presented in FIG. 8D, the survival rate of LB1 in
microparticles formulated with a 1:1 ratio of LMF:NaCas and spray
dried at 80.degree. C. is improved over the survival rate of LB1 in
microparticles having similar water activity but formulated with
NaCas only or with a 1:1 ratio of oil:NaCas.
[0093] It is known that a water activity between 0.11 and 0.23 can
prevent cell death during storage, while water activity above this
range is related to accelerated mortality of probiotics. As seen in
FIG. 8E, the water activity of the present microparticles was found
to be in the acceptable range for maintenance of the survival of
probiotics during storage, over a range of water content
values.
Example 10
Thermal Properties of Emulsions Containing LMF or Vegetable Oil
[0094] Thermal properties of emulsions containing LMF or vegetable
oil in aqueous sodium caseinate (NaCas) solution (10% w/w)
(prepared as described in Example 3) were measured using a
differential scanning calorimeter (DSC, Auto Q20, TA Instruments,
DE, USA). Pure LMF (7 mg) or samples of the emulsion or the
non-emulsified 10% (w/w) aqueous sodium caseinate solution
(control) (50 mg) were weighed and sealed in aluminum pans and
loaded into the DSC. The samples were heated from 0.degree. C. to
80.degree. C. at 1.5.degree. C/min. All measurements were run
against an empty pan and heat flow was recorded as a function of
temperature.
[0095] Results
[0096] Differential scanning calorimetry (DSC) measures the heat
capacity of physical states and the excess heat associated with
transitions that can be induced by temperature change. DSC profiles
for the vegetable oil or LMF emulsions prepared with different
lipid core to sodium caseinate wall ratios are presented in FIGS.
9A and 9B, respectively. Neither endothermic nor exothermic peaks
were observed for the control (10% (w/w) aqueous sodium caseinate
solution) or emulsions made with vegetable oil in the temperature
range from 0.degree. C. to 80.degree. C. (FIG. 9A). As seen in FIG.
9B, however, for the pure LMF sample, there were four peaks in the
temperature range of 0.degree. C. to 80.degree. C., at 5.46.degree.
C., 12.30.degree. C., 21.13.degree. C. and 40.06.degree. C. These
peaks could be associated with the four main fatty acid components
with differing chain lengths that constitute the LMF. For the
emulsions containing LMF and NaCas at different core to wall
ratios, the peak at about 40.06.degree. C. still existed for all
samples. However, the first three peaks seen in the pure LMF sample
were only observed in samples having a high LMF to NaCas ratio,
possibly due to the detection limit of the DSC. With increasing LMF
to NaCas ratios from 0.25 to 1.00, the intensity of all peaks
increased.
[0097] Melting enthalpy (AH) represents the energy required to melt
the crystal fat present in the samples. The AH values of emulsions
with different LMF to NaCas ratios are presented in Table 2.
TABLE-US-00002 TABLE 2 .DELTA.H (J/g) LMF/NaCas (w:w) peak 1, 2, 3
peak 4 Total LMF only 59.66 28.04 87.70 0.25 3.53 1.61 5.14 0.50
7.33 3.52 10.85 0.75 11.39 5.30 16.69 1.00 15.42 7.25 22.67
[0098] As the LMF to NaCas ratio increased from 0.25 to 1.00, AH
increased gradually from 3.53 J/g to 15.42 J/g for the first three
peaks and from 1.61 J/g to 7.25 J/g for the last peak,
respectively. The increased AH in the LMF emulsion samples
suggested that the addition of LMF would provide the emulsion with
endothermic peaks at the temperature around its melting point. The
amount of absorbed heat energy increased with increasing LMF:NaCas
ratio. For the LMF sample with core to wall ratio of 0.25, the
survival rate of LB1 was similar to those of the control and
vegetable oil samples. The total melting enthalpy of the LMF/NaCas
emulsions with different LMF to NaCas ratios was found to
positively correlate with the survival of LB1 after spray drying as
shown in FIG. 10.
[0099] The embodiments described herein are intended to be
illustrative of the present compositions and methods and are not
intended to limit the scope of the present invention. Various
modifications and changes consistent with the description as a
whole and which are readily apparent to the person of skill in the
art are intended to be included. The appended claims should not be
limited by the specific embodiments set forth in the examples, but
should be given the broadest interpretation consistent with the
description as a whole.
* * * * *